Doubled haploid cells, embryos and plants

ABSTRACT

Methods for producing homozygous plants, seeds, and plant cells are provided. Methods of forming haploid tissue and then doubling the chromosomes to form doubled haploid cells are provided. Also provided are methods of transformation.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of application Ser. No. 10/121,200filed Apr. 12, 2002, which claims priority to provisional applicationU.S. Ser. No. 60/285,265 filed Apr. 20, 2001, the disclosures of whichare incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to the field of genetic engineering ofplants and to methods for introducing traits into plants.

BACKGROUND OF THE INVENTION

Many current transformation technologies produce mainly heterozygoustransgenic plants. However, homozygous transgenic plants are basic forproduct development and commercialization of plants. To obtainhomozygous transgenic plants requires several generations ofself-pollination and segregation analysis. This is an inefficient use oflabor and time resources. It would therefore be useful to develop amethod to reduce hand pollination steps normally required to obtain ahomozygous transgenic plant.

DETAILED DESCRIPTION OF THE INVENTION

As used herein “Growth Stimulation Polynucleotides” includepolynucleotides whose encoded products stimulate growth either throughtriggering developmental programs (i.e. embryogenesis, meristemformation, meristem maintenance, etc) or through stimulating the cellcycle.

As used herein “Transformation” includes stable transformation andtransient transformation unless indicated otherwise.

As used herein “Stable Transformation” refers to the transfer of anucleic acid fragment into a genome of a host organism (this includesboth nuclear and organelle genomes) resulting in genetically stableinheritance. In addition to traditional methods, stable transformationincludes the alteration of gene expression by any means includingchimerplasty or transposon insertion.

As used herein “Transient Transformation” refers to the transfer of anucleic acid fragment or protein into the nucleus (or DNA-containingorganelle) of a host organism resulting in gene expression withoutintegration and stable inheritance.

As used herein, “nucleic acid” includes deoxyribonucleotide orribonucleotide polymer, or chimeras thereof, in either single- ordouble-stranded form, and unless otherwise limited, encompasses knownanalogues having the nature of natural nucleotides in that theyhybridize to single-stranded nucleic acids in a manner similar tonaturally occurring nucleotides (e.g., peptide nucleic acids).

As used herein, the term “plant” includes reference to whole plants,plant organs (e.g., leaves, stems, roots, etc.), seeds and plant cellsand progeny of same. “Plant cell”, as used herein includes, withoutlimitation, seeds, suspension cultures, embryos, meristematic regions,callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen,and microspores. The class of plants which can be used in the methods ofthe invention include both monocotyledonous and dicotyledonous plants.

Methods for obtaining homozygous plants, plant cells, and seeds areprovided. Also provided are methods for obtaining haploid embryos andseeds and methods for increasing chromosomal doubling. The methodscomprise contacting haploid cells with a chromosome doubling agent andproviding a growth stimulation protein. The methods also comprisecrossing a selected plant and an inducer line to produce haploid embryosor seeds while providing a growth stimulation polynucleotide. Othermethods comprise crossing a selected plant and an inducer line toproduce a haploid cell, providing a growth stimulation polynucleotide,and treating the haploid cell with a chromosome doubling agent. Alsoprovided are methods for producing transgenic homozygous plants andseeds. The methods comprise transforming a cell from haploid somatictissue such as embryo, meristem, leaf, root, inflorescence, callustissue derived from such tissue, or seed and then contacting thetransformed cell with a chromosome doubling agent. The methods providehomozygous plant cells which can be regenerated into a plant containinghomozygous genes. The methods avoid time consuming crossing methods toobtain a homozygous trait of interest. The methods can be useful forfunctional genomics, such as knock-out analysis, functional analysis ofrecessive genes, gene replacement, gene targeting, transgene stacking,and evaluating lethal versus non-lethal analysis of genes. With thecurrent diploid transformation system, these analyses are verycomplicated and costly. The inventive methods can be used to transformand express recessive genes in T0 plants.

Haploid induction systems have been developed for various plants toproduce haploid tissues, plants and seeds. The haploid induction systemcan produce haploid plants from any genotype by crossing a selected line(as female) with an inducer line. Such inducer lines for maize includeStock 6 (Coe, 1959, Am. Nat. 93:381-382; Sharkar and Coe, 1966, Genetics54:453-464) RWS (Roeber and Geiger 2001, submitted to Crop Science),KEMS (Deimling, Roeber, and Geiger, 1997, Vortr. Pflanzenzuchtg38:203-224), or KMS and ZMS (Chalyk, Bylich & Chebotar, 1994, MNL 68:47;Chalyk & Chebotar, 2000, Plant Breeding 119:363-364), and indeterminategametophyte (ig) mutation (Kermicle 1969 Science 166:1422-1424). Thedisclosures of which are incorporated herein by reference.

Methods for obtaining haploid plants are also disclosed in Kobayashi, M.et al., Journ. of Heredity 71(1):9-14, 1980, Pollacsek, M., Agronomie(Paris) 12(3):247-251, 1992; Cho-Un-Haing et al., Journ. of Plant Biol.,1996, 39(3):185-188; Verdoodt, L., et al., February 1998, 96(2):294-300;Genetic Manipulation in Plant Breeding, Proceedings InternationalSymposium Organized by EUCARPIA, Sep. 8-13, 1985, Berlin, Germany;Chalyk et al., 1994, Maize Genet Coop. Newsletter 68:47; Chalyk, S. T.,1999, Maize Genet. Coop. Newsletter 73:53-54; Coe, R. H., 1959, Am. Nat.93:381-382; Deimling, S. et al., 1997, Vortr. Pflanzenzuchtg 38:203-204;Kato, A., 1999, J. Hered. 90:276-280; Lashermes, P. et al., 1988, Theor.Appl. Genet. 76:570-572 and 76:405-410; Tyrnov, V. S. et al., 1984,Dokl. Akad. Nauk. SSSR 276:735-738; Zabirova, E. R. et al., 1996,Kukuruza I Sorgo N4, 17-19; Aman, M. A., 1978, Indian J. Genet PlantBreed 38:452-457; Chalyk S. T., 1994, Euphytica 79:13-18; Chase, S. S.,1952, Agron. J. 44:263-267; Coe, E. H., 1959, Am. Nat. 93:381-382; Coe,E. H., and Sarkar, K. R., 1964 J. Hered. 55:231-233; Greenblatt, I. M.and Bock, M., 1967, J. Hered. 58:9-13; Kato, A., 1990, Maize Genet.Coop. Newsletter 65:109-110; Kato, A., 1997, Sex. Plant Reprod.10:96-100; Nanda, D. K. and Chase, S. S., 1966, Crop Sci. 6:213-215;Sarkar, K. R. and Coe, E. H., 1966, Genetics 54:453-464; Sarkar, K. R.and Coe, E. H., 1971, Crop Sci. 11:543-544; Sarkar, K. R. and Sachan J.K. S., 1972, Indian J. Agric. Sci. 42:781-786; Kermicle J. L., 1969,Mehta Yeshwant, M. R., Genetics and Molecular Biology, September 2000,23(3):617-622; Tahir, M. S. et al. Pakistan Journal of Scientific andIndustrial Research, August 2000, 43(4):258-261; Knox, R. E. et al.Plant Breeding, August 2000, 119(4):289-298; and U.S. Pat. No. 5,639,951the disclosures of which are incorporate herein by reference.

Somatic haploid cells, haploid embryos, haploid seeds, or haploidseedlings produced from haploid seeds can be treated with a chromosomedoubling agent. Homozygous plants can be regenerated from haploid cellsby contacting the haploid cells, such as embryo cells or callus producedfrom such cells, with chromosome doubling agents, such as colchicine,anti-microtubule herbicides, or nitrous oxide to create homozygousdoubled haploid cells. Treatment of a haploid seed or the resultingseedling generally produces a chimeric plant, partially haploid andpartially doubled haploid. It may be beneficial to nick the seedlingbefore treatment with colchicine. When reproductive tissue containsdoubled haploid cells, then doubled haploid seed is produced.

Haploid embryos, haploid seeds, or somatic haploid cells from a haploidplant can be harvested and transformed by any known means. Transgenichomozygous plants can be regenerated from the transformed cells asdescribed above. Transgenic homozygous seeds can also be produced by themethod described above by treating a haploid seed or the resultingseedling with a chromosome doubling agent and growing the seed toproduce a plant having homozygous seeds.

Methods of chromosome doubling are disclosed in Antoine-Michard, S. etal., Plant Cell, Tissue Organ Cult., Cordrecht, the Netherlands, KluwerAcademic Publishers, 1997, 48(3):203-207; Kato, A., Maize GeneticsCooperation Newsletter 1997, 36-37; and Wan, Y. et al., TAG, 1989, 77:889-892. Wan, Y. et al., TAG, 1991, 81: 205-211. The disclosures ofwhich are incorporated herein by reference. Typical methods involvecontacting the transformed cell with nitrous oxide, anti-microtubuleherbicides, or colchicine.

Polynucleotides or polypeptides involved in growth stimulation or cellcycle stimulation can be used to increase the frequency of haploidembryos produced per ear, increase the recovery of transformed haploidplants, and/or stimulate chromosomal doubling efficiency. The growthstimulation polynucleotide can be provided by either the female or maleparent. The growth stimulation polynucleotide or polypeptide can beprovided by stable or transient transformation.

Polynucleotides whose overexpression has been shown to stimulate thecell cycle include Cyclin A, Cyclin B, Cyclin C, Cyclin D, Cyclin E,Cyclin F, Cyclin G, and Cyclin H; Pin1; E2F; Cdc25; RepA and similarplant viral polynucleotides encoding replication-associated proteins. Inaddition, there are other cell cycle regulatory polynucleotides whoseexpression must be down-regulated to stimulate the cycle and concomitantcell division. These include polynucleotides whose encoded polypeptidesnormally repress the cell cycle, such as Rb, CKI, prohibitin, and wee1.Thus, polynucleotides that encode polypeptides involved in theregulation of the cell cycle in plants can be used in the invention, andinclude cyclins (Doerner (1994) Plant Physiol. 106:823-827.), maize cdc2(Colasanti et al. (1991) PNAS 88:3377-3381), other cdc2 WO 99/53069,cdc25+ (Russell and Nurse (1986) Cell 45:145-153), the geminivirus RepAgene (U.S. Ser. No. 09/257,131), plant E2F (Ramirez-Parra et al. (1999)Nucleic Acid Res. 27:3527-3533 and Sekine et al. (1999) FEBS Lett.460:117-122), Pin1 (Liou et al., 2002, Proc Natl. Acad. Sci. USA99(3):1335-40 and Yao et al., 2001, J. Biol. Chem. 276(17):13517-23),Cyclin D disclosed in WO 00/17364 published Mar. 30, 2000, CKSpolynucleotides disclosed in Ser. No. 99/61619 filed May 19, 1999,Cyclin E polynucleotides disclosed in Ser. No. 09/496,444 filed Feb. 2,2000. Repressors of the cell cycle such as Rb (Grafi et al. (1996) Proc.Natl. Acad. Sci. USA 93(17): 8962-7; Ach et al. (1997) Mol. Cell Biol.17(9):5077-86), CKI (U.S. Ser. No. 01/44038 filed Nov. 6, 2001),prohibitin (WO 00/15818), and wee1 (disclosed in WO 00/37645) genes canbe used in the practice of the invention. The disclosures of which areherein incorporated by reference.

Examples of plant virus replicase polynucleotide sources suitable forgrowth stimulation (i.e. stimulation of S-phase in the cell cycle)include wheat dwarf virus, maize streak virus, tobacco yellow dwarfvirus, tomato golden mosaic virus, abutilon mosaic virus, cassava mosaicvirus, beet curly top virus, bean dwarf mosaic virus, bean golden mosaicvirus, chloris striate mosaic virus, digitaria streak virus, miscanthusstreak virus, maize streak virus, panicum streak virus, potato yellowmosaic virus, squash leaf curl virus, sugarcane streak virus, tomatogolden mosaic virus, tomato leaf curl virus, tomato mottle virus,tobacco yellow dwarf virus, tomato yellow leaf curl virus, Africancassava mosaic virus, and the bean yellow dwarf virus. Replicase fromthe wheat dwarf virus has been sequenced and functionally characterized.Replicase binds to a well-characterized binding motif on the Rb protein(Xie et al., The EMBO J., 14(16):4073-4082, 1995; Orozco et al., J.Biol. Chem., 272(15):9840-9846, 1997; Timmermans et al., Annual ReviewPlant Physiology. Plant Mol. Biol, 45:79-112, 1994; Stanley, Geneticsand Development 3:91-96, 1996; Davies et al., Geminivirus Genomes,Chapter 2, and Gutierrez, Plant Biology 1:492-497, 1998). Other growthstimulation (S-phase stimulating) polynucleotides suitable for useinclude viral cell cycle modulator proteins such as CLINK (Aronson et alJournal of Virology 74:2968-2972, 2000). Examples of other viral sourcesfor this type of protein include banana bunchy top virus, milk vetchdwarf virus, subterranean clover stunt virus Ageratum yellow vein virusand other representatives of plant nanoviruses. The disclosures of theseitems are incorporated herein by reference.

Growth stimulation polynucleotides include polynucleotides whoseoverexpression stimulates growth through triggering developmentalprograms include such examples as SERK, Lec1, Lec2, WUS, FUS3, ABI3(Vp1), BMN3, ANT, and members of the Knotted family, such as Kn1, STM,OSH1, and SbH1; cytokinin genes such as IPT, TZS, CKI-1; and genes thatproduce growth stimulating peptides such as PSK. Also, genes whoseencoded products repress specific plant developmental programs can bedown-regulated to stimulate growth, such as the gene PICKLE, that whendown-regulated results in embryogenic growth. Thus, these genes usefulin the present invention include the Kn1 family of genes disclosed inVollbrecht et al., Nature 350:241-243, 1991; Sentoku et al., Develop.Biol. 220:358-364, 2000 and Sinha et al., 1993, Genes Dev 7(5):787-95,WUSCHEL or WUS genes found in Mayer et al., Cell 95:805-815, 1998;Lenhard et al., Cell 105(6):805-14; and Laux et al. 1996, Development122(1):87-96, Lec1 polynucleotides disclosed in U.S. Ser. No. 99/26514filed Nov. 9, 1999, SERK polynucleotides disclosed in Schmidt et al.1997, Development 124(10):2049-62 and Baudino et al. 2001, Planta213(1):1-10, Babyboom (BMN3) polynucleotides disclosed in EP1057891(A1), LEC2 polynucleotides disclosed in Stone et al. 2001, Proc NatlAcad Sci U S A 98(20):11806-11, FUS3 polynucleotides disclosed inNambara et al. 2000, Dev Biol 220(2):412-23 and Vicient et al., 2000, JExp Bot 51(347):995-1003, STM polynucleotides disclosed in Endrizzi etal. 1996, Plant J 10(6):967-79 and Long et al. 1996, Nature379(6560):66-9. 8, ANT (Aintegument) polynucleotides disclosed inMizukami, 2001, Curr Opin Plant Biol 4(6):533-9; Nole-Wilson S, Krizek BA., 2000, Nucleic Acids Res 28(21):4076-82; Mizukami Y, Fischer R L.,2000, Proc Natl Acad Sci USA 97(2):942-7; and Krizek B A., 1999, DevGenet 25(3):224-36, ABI3 polynucleotides disclosed in Suzuki et al.2001, Plant J 28(4):409-18; Rohde et al., 2000, Trends Plant Sci5(10):418-9; Parcy et al., 1997, Plant Cell 9(8):1265-77; Parcy F,Giraudat J., 1997, Plant J 11(4):693-702, and PICKLE (Ogas et al., PNAS96:13839-13844, 1999). Genes that stimulate growth by encoding productsinvolved in the synthesis of growth stimulating hormones (IPT, TZS),that confer independence from a hormone (CKI1) or in which the peptideitself is a growth stimulating hormone (PSK). Thus, such genes can beused in the present invention and include the IPT gene of Agrobacteriumtumefaciens (Strabala et al. (1989) Mol. Gen. Genet. 216:388-394,Bonnard et al. (1989) Mol. Gen. Genet. 216:428-438, DDBJ/EMBL/GenBank),TZS (Beaty et al. (1986) Mol. Gen. Genet. 203:274-280, Akiyoshi et al.(1985) Nucleic Acids Res. 13:2773-2788, Regier et al. (1989) NucleicAcids Res. 17:8885), CKI1 (Kakimoto (1996) Science 274:982-985), andPSKα (Yang et al. (1999) PNAS 96:13560-13565). The disclosures of theabove are incorporated herein by reference.

As discussed above, growth stimulation polynucleotides (or polypeptides)can be used to increase chromosomal doubling in haploid plant tissues(callus, seeds, seedlings etc.) with the methods described herein. Thefrequency of doubled haploids can be increased several fold. The growthstimulation polynucleotides can be introduced into the male or femaleparent. Introducing the growth stimulation polynucleotides into thematernal parent will result in a plant homozygous for the growthstimulation polynucleotide. If the growth stimulation polynucleotide isintroduced into the paternal parent (the inducer line) the growthstimulation polynucleotide would be present in the endosperm, but not inthe embryo. This can result in increased vigor of the haploid embryo.

After successful doubling of the haploid chromosomes, it may bedesirable to remove the above growth stimulation polynucleotides. Thiscan be accomplished by using various methods of gene excision, such asthose described below including the use of recombination sites andrecombinases.

In another aspect the inducer line may contain a scorable marker gene,for example colored markers in the endosperm, embryo or stem. Suchmarkers include GUS (U.S. Pat. No. 5,599,670 and U.S. Pat. No.5,432,081), GFP (U.S. Pat. No. 6,146,826; U.S. Pat. No. 5,491,084; andWO 97/41228), luciferase (U.S. Pat. No. 5,674,713 and Ow et al. 1986Science 234 (4778) 856-859), CRC (Ludwig et al., 1990) other anthocyaningenes such as A, C, R-nj, etc. and others known in the art. Thedisclosures of which are incorporated herein by reference. When theinducer line is crossed with the selected line, the resulting haploidseeds will have colored endosperm with colorless embryo. Some linesalready contain a color marker. For various reasons it may be desirableto express the marker gene in the embryo. In particular, it may bedesirable to express the marker gene in the early stage of development,about 8-15 days after pollination using an appropriate promoter such asan oleosin or a Lec1 promoter. Marker negative embryos are then selectedto obtain haploid embryos. This method provides the advantage ofobtaining haploid embryos without marker genes.

The methods of the invention can be practiced with any plant. Suchplants include but are not limited to maize, soybean, oilseed Brassica,alfalfa, rice, rye, sorghum, sunflower, tobacco, potato, peanuts,cotton, sweet potato, cassava, sugar beets, tomato, oats, barley, andwheat.

Genes of interest are reflective of the commercial markets and interestsof those involved in the development of the crop. Crops and markets ofinterest change, and as developing nations open up world markets, newcrops and technologies will emerge also. In addition, as ourunderstanding of agronomic traits and characteristics such as yield andheterosis increases, the choice of genes for transformation will changeaccordingly. It is also understood that two or more genes may beintroduced into a plant.

General categories of genes of interest include for example, those genesinvolved in information, such as zinc fingers, those involved incommunication, such as kinases, and those involved in housekeeping, suchas heat shock proteins. More specific categories of transgenes, forexample, include genes encoding agronomic traits, insect resistance,disease resistance, herbicide resistance, sterility, graincharacteristics, and commercial products. Genes of interest also includethose involved in oil, starch, carbohydrate, or nutrient metabolism aswell as those affecting for example kernel size, sucrose loading, andthe like. The quality of grain is reflected in traits such as levels andtypes of oils, saturated and unsaturated, quality and quantity ofessential amino acids, and levels of cellulose.

Grain traits such as oil, starch, and protein content can be geneticallyaltered. Modifications include increasing the content of oleic acid,saturated or unsaturated oils, increasing levels of lysine and sulfur,providing essential amino acids, and also modification of starch.Hordothionin protein modifications are described in U.S. Pat. No.5,990,389 issued Nov. 23, 1999, U.S. Pat. No. 5,885,801 issued Mar. 23,1999, U.S. Pat. No. 5,885,802 issued Mar. 23, 1999 and U.S. Pat. No.5,703,409. Another example is lysine and/or sulfur rich seed proteinencoded by the soybean 2S albumin described in U.S. Pat. No. 5,850,016issued Dec. 15, 1998, and the chymotrypsin inhibitor from barley,Williamson et al. (1987) Eur. J. Biochem. 165:99-106. The disclosures ofthe above are herein incorporated by reference.

Derivatives of the coding sequences can be made by site-directedmutagenesis to increase the level of preselected amino acids in theencoded polypeptide. For example, the gene encoding the barley highlysine polypeptide (BHL) is derived from barley chymotrypsin inhibitorWO 98/20133 the disclosure of which is incorporated herein by reference.Other proteins include methionine-rich plant proteins such as from corn(Pedersen et al. (1986) J. Biol. Chem. 261:6279; Kirihara et al. (1988)Gene 71:359; and rice (Musumura et al. (1989) Plant Mol. Biol. 12:123).The disclosures of which are incorporated herein by reference. Othergenes encode latex, Floury 2, growth factors, seed storage factors, andtranscription factors.

Insect resistance genes may encode resistance to pests that have greatyield drag such as rootworm, cutworm, European Corn Borer, and the like.Such genes include, for example Bacillus thuringiensis toxic proteingenes (U.S. Pat. Nos. 5,366,892; 5,747,450; 5,737,514; 5,723,756;5,593,881; Geiser et al. (1986) Gene 48:109); lectins (Van Damme et al.(1994) Plant Mol. Biol. 24:825); and the like. Genes encoding diseaseresistance traits include detoxification genes, such as againstfumonisin (U.S. Pat. No. 5,792,931, issued Aug. 11, 1998); avirulence(avr) and disease resistance genes (Jones et al. (1994) Science 266:789;Martin et al. (1993) Science 262:1432; Mindrinos et al. (1994) Cell78:1089); and the like.

Herbicide resistance traits may include genes coding for resistance toherbicides that act to inhibit the action of acetolactate synthase(ALS), in particular the sulfonylurea-type herbicides (e.g., theacetolactate synthase (ALS) gene containing mutations leading to suchresistance, in particular the S4 and/or Hra mutations), genes coding forresistance to herbicides that act to inhibit action of glutaminesynthase, such as phosphinothricin or basta (e.g., the bar gene), orother such genes known in the art. The bar gene encodes resistance tothe herbicide basta, the nptII gene encodes resistance to theantibiotics kanamycin and geneticin, and the ALS gene encodes resistanceto the herbicide chlorsulfuron. Glyphosate tolerance can be obtainedfrom the EPSPS gene.

Sterility genes can also be encoded in an expression cassette andprovide an alternative to physical detasseling. Examples of genes usedin such ways include male tissue-preferred genes and genes with malesterility phenotypes such as QM, described in U.S. Pat. No. 5,583,210.Other genes include kinases and those encoding compounds toxic to eithermale or female gametophytic development.

Commercial traits can also be encoded on a gene or genes that couldincrease for example, starch for ethanol production, or provideexpression of proteins. Another commercial use of transformed plants isthe production of polymers and bioplastics such as described in U.S.Pat. No. 5,602,321 issued Feb. 11, 1997. Genes such as B-Ketothiolase,PHBase (polyhydroxybutyrate synthase) and acetoacetyl-CoA reductase (seeSchubert et al. (1988) J. Bacteriol. 170:5837-5847) facilitateexpression of polyhyroxyalkanoates (PHAs). Genes of medicinal andpharmaceutical uses, such as that encoding avidin and vaccines orproteins produced utilizing plants as factories are also contemplated aspart of this invention.

It is recognized that the present invention contemplates the use ofvarious gene targeting methods. Insertion, excision or recombinationsites for use in the invention are known in the art and include FRT orlox sites (see, for example, Schlake et al. (1994) Biochemistry33:12746-12751; Huang et al. (1991) Nucleic Acids Res. 19:443-448;Sadowski (1995) Prog. Nuc. Acid Res. Mol. Bio. 51:53-91; Cox (1989)Mobile DNA, ed. Berg and Howe (American Society of Microbiology,Washington D.C.), pp. 116-670; Dixon et al. (1995) 18:449-458; Umlauf etal. (1988) EMBO J. 7:1845-1852; Buchholz et al. (1996) Nucleic AcidsRes. 24:3118-3119; Kilby et al. (1993) Trends Genet. 9:413-421; Roseanneet al. (1995) Nat. Med. 1:592-594; Albert et al. (1995) Plant J.7:649-659; Bailey et al. (1992) Plant Mol. Biol. 18:353-361; Odell etal. (1990) Mol. Gen. Genet. 223:369-378; and Dale et al. (1991) Proc.Natl. Acad. Sci. USA 88:10558-105620; lox (Albert et al. (1995) Plant J.7:649-659; Qui et al. (1994) Proc. Natl. Acad. Sci. USA 91:1706-1710;Stuurman et al. (1996) Plant Mol. Biol. 32:901-913; Odell et al. (1990)Mol. Gen. Genet. 223:369-378; Dale et al. (1990) Gene, 91:79-85; andBayley et al. (1992) Plant Mol. Biol. 18:353-361); U.S. Pat. No.5,658,772; U.S. Pat. No. 4,959,317; U.S. Pat. No. 6,110,736. Suchrecombination sites in the presence of a compatible recombinase allowfor the targeted integration of one or more nucleotide sequences ofinterest into the plant genome. It is recognized that variations oftargeted insertion can also be practiced with the invention. See forexample WO 99/25821; WO 99/25855; WO 99/25840; WO 99/25853. Thedisclosures of the above are herein incorporated by reference.

Where appropriate, the nucleotide sequences of interest may be optimizedfor increased expression in the plant. Where mammalian, yeast, orbacterial genes are used in the invention, they can be synthesized usingplant-preferred codons for improved expression. It is recognized thatfor expression in monocots, dicot genes can also be synthesized usingmonocot-preferred codons. Methods are available in the art forsynthesizing plant-preferred genes. See, for example, U.S. Pat. Nos.5,380,831, 5,436, 391, and Murray et al. (1989) Nucleic Acids Res.17:477-498, herein incorporated by reference.

The plant-preferred codons may be determined from the codons utilizedmore frequently in the proteins expressed in the recipient plant ofinterest. It is recognized that monocot-or dicot-preferred sequences maybe constructed as well as plant-preferred sequences for particular plantspecies. See, for example, EPA 0359472; EPA 0385962; WO 91/16432; Perlaket al. (1991) Proc. Natl. Acad. Sci. USA 88:3324-3328; and Murray et al.(1989) Nucleic Acids Res. 17:477-498; U.S. Pat. Nos. 5,380,831 and5,436,391; and the like, herein incorporated by reference. It is furtherrecognized that all or any part of the gene sequence may be optimized orsynthetic. That is, fully optimized or partially optimized sequences mayalso be used.

Additional sequence modifications are known to enhance gene expressionin a cellular host and can be used in the invention. These includeelimination of sequences encoding spurious polyadenylation signals,exon-intron splice site signals, transposon-like repeats, and other suchwell-characterized sequences, which may be deleterious to geneexpression. The G-C content of the sequence may be adjusted to levelsaverage for a given cellular host, as calculated by reference to knowngenes expressed in the host cell. When possible, the sequence may bemodified to avoid predicted hairpin secondary mRNA structures.

In one example, where a DNA construct comprising a compatiblerecombinase gene is to be used for targeted integration of a nucleotidesequence of interest into a target site within a chromosome of interest,the nucleotide sequence encoding the compatible recombinase may beconstructed with plant-preferred codons. More particularly, where thegene encodes a FLP recombinase, for example, the FLP gene sequence maybe constructed using plant-preferred codons to obtain an FLP recombinasethat is optimized for expression in the plant, WO 99/27077, thedisclosure of which is incorporated herein by reference.

The nucleotide sequences of interest may be utilized in an expressioncassette. Generally the nucleotide sequence of interest is operablylinked with a functional promoter, and in most instances a terminationregion. There are various ways to achieve the expression cassette withinthe practice of the invention. In one embodiment of the invention, thenucleotide sequence of interest is transferred or inserted into thegenome as an expression cassette. Alternatively, the nucleotide sequencemay be inserted into a site within the genome that is 3′ to a promoterregion. In this latter instance, the insertion of the coding sequence 3′to the promoter region is such that a functional expression cassette isachieved upon integration.

For convenience, the nucleotide sequences of interest are generallyprovided in expression cassettes for expression in the plant. Thecassette will include 5′ and 3′ regulatory sequences operably linked toa nucleotide sequence of interest. By “operably linked” is intended afunctional linkage between a promoter and a second sequence, wherein thepromoter sequence initiates and mediates transcription of the nucleicacid sequence corresponding to the second sequence. The cassette mayadditionally contain at least one additional gene or nucleotide sequenceof interest to be cotransformed into the plant. Thus, each nucleic acidsequence will be operably linked to 5′ and 3′ regulatory sequences.Alternatively, the additional gene(s) or nucleotide sequence(s) can beprovided on multiple expression cassettes.

The construction of such expression cassettes which can be employed inconjunction with the present invention is well known to those of skillin the art in light of the present disclosure. See, e.g., Sambrook etal., Molecular Cloning: A Laboratory Manual; Cold Spring Harbor, N.Y.;(1989); Gelvin et al., Plant Molecular Biology Manual (1990); PlantBiotechnology: Commercial Prospects and Problems, eds. Prakash et al.,Oxford & IBH Publishing Co.; New Delhi, Ind.; (1993); and Heslot et al.,Molecular Biology and Genetic Engineering of Yeasts; CRC Press, Inc.,USA; (1992); each disclosure incorporated herein by reference.

For example, plant expression vectors may include (1) a cloned plantgene under the transcriptional control of 5′ and 3′ regulatory sequencesand (2) a dominant selectable marker. Such plant expression vectors mayalso contain, if desired, a promoter regulatory region (e.g., oneconferring inducible, constitutive, environmentally- ordevelopmentally-regulated, or cell- or tissue-specific/selectiveexpression), a transcription initiation start site, a ribosome bindingsite, an RNA processing signal, a transcription termination site, and/ora polyadenylation signal. Such an expression cassette is generallyprovided with a plurality of restriction sites for insertion of thenucleotide sequence of interest that is to be under the transcriptionalregulation of the regulatory regions.

The expression cassette may additionally contain selectable markergenes. The marker gene confers a selectable phenotype on plant cells.Usually, the selectable marker gene will encode antibiotic or herbicideresistance. Suitable genes include those coding for resistance to theantibiotics spectinomycin and streptomycin (e.g., the aada gene), thestreptomycin phosphotransferase (SPT) gene coding for streptomycinresistance, the neomycin phosphotransferase (NPTII) gene encodingkanamycin or geneticin resistance, the hygromycin phosphotransferase(HPT) gene coding for hygromycin resistance.

Suitable genes coding for resistance to herbicides include those whichact to inhibit the action of acetolactate synthase (ALS), in particularthe sulfonylurea-type herbicides (e.g., the acetolactate synthase (ALS)gene containing mutations leading to such resistance in particular theS4 and/or Hra mutations), those which act to inhibit action of glutaminesynthase, such as phosphinothricin or basta (e.g., the bar gene), orother such genes known in the art. The bar gene encodes resistance tothe herbicide basta and the ALS gene encodes resistance to the herbicidechlorsulfuron.

Selectable marker genes for the selection of transformed cells ortissues are disclosed in the following publications. See generally,Yarranton (1992) Curr. Opin. Biotech. 3:506-511; Christopherson et al.(1992) Proc. Natl. Acad. Sci. USA 89:6314-6318; Yao et al. (1992) Cell71:63-72; Reznikoff (1992) Mol. Microbiol. 6:2419-2422; Barkley et al.(1980) Operon, pp. 177-220; Hu et al. (1987) Cell 48:555-566; Brown etal. (1987) Cell 49:603-612; Figge et al. (1988) Cell 52:713-722;Deuschle et al. (1989) Proc. Natl. Acad. Sci. USA 86:5400-5404; Fuerstet al. (1989) Proc. Natl. Acad. Sci. USA 86:2549-2553; Deuschle et al.(1990) Science 248:480-483; M. Gossen (1993) Ph.D dissertation,University of Heidelberg; Reines et al. (1993) Proc. Natl. Acad. Sci.USA 90:1917-1921; Labow et al. (1990) Mol. Cell Bio. 10:3343-3356;Zambretti et al. (1992) Proc. Natl. Acad. Sci. USA 89:3952-3956; Baim etal. (1991) Proc. Natl. Acad. Sci. USA 88:5072-5076; Wyborski et al.(1991) Nucleic Acids Res. 19:4647-4653; Hillenand-Wissman (1989) Topicsin Mol. and Struc. Biol. 10:143-162; Degenkolb et al. (1991) Antimicrob.Agents Chemother. 35:1591-1595; Kleinschnidt et al. (1988) Biochemistry27:1094-1104; Gatz et al. (1992) Plant J. 2:397-404; A. L. Bonin (1993)Ph.D. dissertation, University of Heidelberg; Gossen et al. (1992) Proc.Natl. Acad. Sci. USA 89:5547-5551; Oliva et al. (1992) Antimicrob.Agents Chemother. 36:913-919; Hlavka et al. (1985) Handbook of Exp.Pharmacology 78; Gill et al. (1988) Nature 334:721-724. Such disclosuresare herein incorporated by reference.

The expression cassette will generally include in the 5′-3′ direction oftranscription, a transcriptional and translational initiation region, anucleotide sequence of interest, and a transcriptional and translationaltermination region functional in plants. The transcriptional initiationregion, the promoter, may be native or analogous or foreign orheterologous to the plant host. Additionally, the promoter may be thenatural sequence or alternatively a synthetic sequence. By “foreign” isintended that the transcriptional initiation region is not found in thenative plant into which the transcriptional initiation region isintroduced.

While it may be preferable to express the nucleotide sequences ofinterest using heterologous promoters, the native promoter sequences maybe used. Such constructs would change expression levels of any proteinencoded by a nucleotide sequence of interest in the plant or plant cell.Thus, the phenotype of the plant or plant cell is altered.

Constitutive, tissue-preferred or inducible promoters can be employed.Examples of constitutive promoters include the cauliflower mosaic virus(CaMV) 35S transcription initiation region, the 1′- or 2′-promoterderived from T-DNA of Agrobacterium tumefaciens, the actin promoter, theubiquitin promoter, the histone H2B promoter (Nakayama et al., 1992,FEBS Lett 30:167-170), the Smas promoter, the cinnamyl alcoholdehydrogenase promoter (U.S. Pat. No. 5,683,439), the Nos promoter, thepEmu promoter, the rubisco promoter, the GRP1-8 promoter, and othertranscription initiation regions from various plant genes known in theart.

Examples of inducible promoters are the Adh1 promoter which is inducibleby hypoxia or cold stress, the Hsp70 promoter which is inducible by heatstress, the PPDK promoter which is inducible by light, the In2 promoterwhich is safener induced, the ERE promoter which is estrogen induced andthe Pepcarboxylase promoter which is light induced.

Examples of promoters under developmental control include promoters thatinitiate transcription preferentially in certain tissues, such asleaves, roots, fruit, seeds, or flowers. An exemplary promoter is theanther specific promoter 5126 (U.S. Pat. Nos. 5,689,049 and 5,689,051).Examples of seed-preferred promoters include, but are not limited to, 27kD gamma zein promoter and waxy promoter, Boronat, A., Martinez, M. C.,Reina, M., Puigdomenech, P. and Palau, J.; Isolation and sequencing of a28 kD glutelin-2 gene from maize: Common elements in the 5′ flankingregions among zein and glutelin genes; Plant Sci. 47:95-102 (1986) andReina, M., Ponte, I., Guillen, P., Boronat, A. and Palau, J., Sequenceanalysis of a genomic clone encoding a Zc2 protein from Zea mays W64 A,Nucleic Acids Res. 18(21):6426 (1990). See the following site relatingto the waxy promoter: Kloesgen, R. B., Gierl, A., Schwarz-Sommer, Z. S.and Saedler, H., Molecular analysis of the waxy locus of Zea mays, Mol.Gen. Genet. 203:237-244 (1986). The disclosures of each of these areincorporated herein by reference. The barley or maize Nuc1 promoter, themaize Cim 1 promoter or the maize LTP2 promoter can be used topreferentially express in the nucellus. See for example U.S. Ser. No.60/097,233 filed Aug. 20, 1998 the disclosure of which is incorporatedherein by reference.

Either heterologous or non-heterologous (i.e., endogenous) promoters canbe employed to direct expression of the nucleic acids of the presentinvention. These promoters can also be used, for example, in expressioncassettes to drive expression of antisense nucleic acids to reduce,increase, or alter concentration and/or composition of the proteins ofthe present invention in a desired tissue.

The termination region is optional and may be native with thetranscriptional initiation region, may be native with the operablylinked DNA sequence of interest, or may be derived from another source.Convenient termination regions are available from the potato proteinaseinhibitor (PinII) gene or the Ti-plasmid of A. tumefaciens, such as theoctopine synthase and nopaline synthase termination regions. See alsoGuerineau et al. (1991) Mol. Gen. Genet. 262:141-144; Proudfoot (1991)Cell 64:671-674; Sanfacon et al. (1991) Genes Dev. 5:141-149; Mogen etal. (1990) Plant Cell 2:1261-1272; Munroe et al. (1990) Gene 91:151-158;Ballas et al. (1989) Nucleic Acids Res. 17:7891-7903; and Joshi et al.(1987) Nucleic Acid Res. 15:9627-9639. The disclosures of the above areherein incorporated by reference.

The expression cassettes may additionally contain 5′ leader sequences inthe expression cassette construct. Such leader sequences can act toenhance translation. Translation leaders are known in the art andinclude: picornavirus leaders, for example, EMCV leader(Encephalomyocarditis 5′ noncoding region) (Elroy-Stein et al. (1989)PNAS USA 86:6126-6130); potyvirus leaders, for example, TEV leader(Tobacco Etch Virus) (Allison et al. (1986); MDMV leader (Maize DwarfMosaic Virus); Virology 154:9-20), and human immunoglobulin heavy-chainbinding protein (BiP), (Macejak et al. (1991) Nature 353:90-94);untranslated leader from the coat protein mRNA of alfalfa mosaic virus(AMV RNA 4) (Jobling et al. (1987) Nature 325:622-625); tobacco mosaicvirus leader (TMV) (Gallie et al. (1989) in Molecular Biology of RNA,ed. Cech (Liss, N.Y.), pp. 237-256); and maize chlorotic mottle virusleader (MCMV) (Lommel et al. (1991) Virology 81:382-385). See also,Della-Cioppa et al. (1987) Plant Physiol. 84:965-968. The disclosures ofthe above are herein incorporated by reference. Other methods known toenhance translation can also be utilized, for example, introns, and thelike.

In preparing the expression cassette, the various DNA fragments may bemanipulated, so as to provide for the DNA sequences in the properorientation and, as appropriate, in the proper reading frame. Towardthis end, adapters or linkers may be employed to join the DNA fragmentsor other manipulations may be involved to provide for convenientrestriction sites, removal of superfluous DNA, removal of restrictionsites, or the like. For this purpose, in vitro mutagenesis, primerrepair, restriction, annealing, resubstitutions, e.g., transitions andtransversions, may be involved.

Once the appropriate plant cells are produced, the nucleotide sequencesof interest can be introduced into the plant cells by any method knownin the art. In this manner, genetically modified plants, plant cells,plant tissue, seed, and the like can be obtained. Transformationprotocols as well as protocols for introducing nucleotide sequences intoplants may vary depending on the type of plant or plant cell, i.e.,monocot or dicot, targeted for transformation. Suitable methods ofintroducing nucleotide sequences into plant cells and subsequentinsertion into the plant genome include microinjection (Crossway et al.(1986) Biotechniques 4:320-334), electroporation (Riggs et al. (1986)Proc. Natl. Acad. Sci. USA 83:5602-5606, Agrobacterium-mediatedtransformation (Townsend et al., U.S. Pat. No. 5,563,055), direct genetransfer (Paszkowski et al. (1984) EMBO J. 3:2717-2722), and ballisticparticle acceleration (see, for example, Sanford et al., U.S. Pat. No.4,945,050; Tomes et al. (1995) “Direct DNA Transfer into Intact PlantCells via Microprojectile Bombardment,” in Plant Cell, Tissue, and OrganCulture: Fundamental Methods, ed. Gamborg and Phillips (Springer-Verlag,Berlin); and McCabe et al. (1988) Biotechnology 6:923-926). Also seeWeissinger et al. (1988) Ann. Rev. Genet. 22:421-477; Sanford et al.(1987) Particulate Science and Technology 5:27-37 (onion); Christou etal. (1988) Plant Physiol. 87:671-674 (soybean); McCabe et al. (1988)Bio/Technology 6:923-926 (soybean); Finer and McMullen (1991) In VitroCell Dev. Biol. 27P:175-182 (soybean); Singh et al. (1998) Theor. Appl.Genet. 96:319-324 (soybean); Datta et al. (1990) Biotechnology 8:736-740(rice); Klein et al. (1988) Proc. Natl. Acad. Sci. USA 85:4305-4309(maize); Klein et al. (1988) Biotechnology 6:559-563 (maize); Tomes,U.S. Pat. No. 5,240,855; Buising et al., U.S. Pat. Nos. 5,322,783 and5,324,646; Tomes et al. (1995) “Direct DNA Transfer into Intact PlantCells via Microprojectile Bombardment,” in Plant Cell, Tissue, and OrganCulture: Fundamental Methods, ed. Gamborg (Springer-Verlag, Berlin)(maize); Klein et al. (1988) Plant Physiol. 91:440-444 (maize); Fromm etal. (1990) Biotechnology 8:833-839 (maize); Hooykaas-Van Slogteren etal. (1984) Nature (London) 311:763-764; Bowen et al., U.S. Pat. No.5,736,369 (cereals); Bytebier et al. (1987) Proc. Natl. Acad. Sci. USA84:5345-5349 (Liliaceae); De Wet et al. (1985) in The ExperimentalManipulation of Ovule Tissues, ed. Chapman et al. (Longman, N.Y.), pp.197-209 (pollen); Kaeppler et al. (1990) Plant Cell Reports 9:415-418and Kaeppler et al. (1992) Theor. Appl. Genet. 84:560-566(whisker-mediated transformation); D'Halluin et al. (1992) Plant Cell4:1495-1505 (electroporation); Li et al. (1993) Plant Cell Reports12:250-255 and Christou and Ford (1995) Annals of Botany 75:407-413(rice); Ishida et al. (1996) Nature Biotechnology 14:745-750; US5,731,179; U.S. Pat. No. 5,591,616; U.S. Pat. No. 5,641,664; and U.S.Pat. No. 5,981,840 (maize via Agrobacterium tumefaciens); thedisclosures of which are herein incorporated by reference.

In planta Agrobacterium transformation is disclosed in the following:Bechtold, N., J. Ellis, G. Pelletier (1993) C. R., Acad Sci Paris LifeSci 316: 1194-1199; Bechtold, N., B. et al. (2000) Genetics155:1875-1887; Bechtold, N. and G. Pelletier (1998) Methods Mol Biol.82:259-266; Chowrira, G. M., V. Akella, and P. F. Lurquin. (1995) Mol.Biotechnol. 3:17-23; Clough, S. J., and A. F. Bent. (1998) Plant J.16:735-743; Desfeux, C., S. J. Clough, and A. F. Bent. (2000) PlantPhysiol. 123: 895-904; Feldmann, K. A., and M. D. Marks. (1987) Mol.Gen. Genet. 208:1-9; Hu C.-Y., and L. Wang. (1999) In Vitro Cell Dev.Biol.-Plant 35:417-420; Katavic, V. G. W. Haughn, D. Reed, M. Martin, L.Kunst (1994) Mol. Gen. Genet. 245: 363-370; Liu, F., et al. (1998) ActaHort 467:187-192; Mysore, K. S., C. T. Kumar, and S. B. Gelvin. (2000)Plant J. 21:9-16; Touraev, A., E. Stoger, V. Voronin, and E.Heberle-Bors. (1997) Plant J. 12:949-956; Trieu, A. T. et al. (2000)Plant J. 22:531-541; Ye, G. N. et al. (1999) Plant J. 19:249-257; Zhang,J U. et al. (2000) Chem Biol. 7:611-621. The disclosures of the aboveare herein incorporated by reference.

Various types of plant tissue can be used for transformation such asembryo cells, meristematic cells, leaf cells, or callus cells derivedfrom embryo, leaf or meristematic cells. However, anytransformation-competent cell or tissue can be used. Various methods forincreasing transformation frequency may also be employed. Such methodsare disclosed in WO 99/61619; WO 00/17364; WO 00/28058; WO 00/37645;U.S. Ser. No. 09/496,444; WO 00/50614; U.S. Ser. No. 01/44038; and WO02/04649. The disclosures of the above are herein incorporated byreference.

The transformed cells can be contacted with a chromosome doubling agentsuch as colchicine, anti-microtubule herbicide, or nitrous oxide in anamount sufficient to produce a doubled haploid cell. The transformedcell can be contacted with the doubling agent before, during, or afterthe plant regeneration step. Haploid seeds and seedlings produced by theseeds can also be treated with the doubling agent.

Once the DNA sequence of interest has been introduced into tissue fromthe plant, transformed cells are selected and transgenic plantsregenerated using methods well known in the art. See, for example,McCormick et al. (1986) Plant Cell Reports 5:81-84; and U.S. Pat. No.5,981,840. Transformed plant cells which are derived by any of the abovetransformation techniques can be cultured to regenerate a whole plantwhich possesses the transformed genotype. Such regeneration techniquesoften rely on manipulation of certain phytohormones in a tissue culturegrowth medium, typically relying on a biocide and/or herbicide markerthat has been introduced together with a polynucleotide of the presentinvention. For transformation and regeneration of maize see, Gordon-Kammet al., The Plant Cell 2:603-618 (1990). Other methods of regeneratingplants can be achieved as described by Horsch et al., Science227:1229-1231 (1985) and Fraley et al., Proc. Natl. Acad. Sci. USA80:4803 (1983). This procedure typically produces shoots within two tofour weeks and these transformant shoots are then transferred to anappropriate root-inducing medium containing the selective agent and anantibiotic to prevent bacterial growth. The disclosures of the above areherein incorporated by reference.

Regeneration can also be obtained from plant callus, explants, organs,or parts thereof. Such regeneration techniques are described generallyin Klee et al., Ann. Rev. of Plant Phys. 38:467-486 (1987). Theregeneration of plants from either single plant protoplasts or variousexplants is well known in the art. See, for example, Methods for PlantMolecular Biology, A. Weissbach and H. Weissbach, eds., Academic Press,Inc., San Diego, Calif. (1988). For maize cell culture and regenerationsee generally, The Maize Handbook, Freeling and Walbot, Eds., Springer,N.Y. (1994); Corn and Corn Improvement, 3rd edition, Sprague and DudleyEds., American Society of Agronomy, Madison, Wis. (1988). Thedisclosures of the above are herein incorporated by reference.

These regenerated transgenic plants may then be grown to maturity andsexually crossed with the same transformed strain (“selfed”), or“backcrossed” with another plant chosen to obtain transgenic plantshaving desired characteristics. Alternatively, the regeneratedtransgenic plants may be used to “introgress” the nucleotide sequence ofinterest into another genetic line of the same plant species or into agenetic line of another closely related plant species.

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

The following examples are offered by way of illustration and not by wayof limitation.

EXAMPLES Example 1 Transformation and Regeneration of Transgenic MaizePlants

Seeds from any maize genotype are planted and the resulting plants areused as female parent plants (pollen receivers). Seeds from haploidinducer lines, such as Stock 6, RWS, KEMS, KMS or ZMS, are planted andthe resulting plants are used as male parent plants (pollen donors). Theears of the female parent plants are shoot-bagged before silk emergence.The silks of the ears on the plants of the female parent plants arepollinated with viable pollen grains collected from the anthers of themale parent plants (haploid inducer plants). This pollination iscontrolled by the method used regularly in maize breeding program toavoid any foreign pollen contamination. The pollination method resultsin the production of a frequency of about 5-12% of haploid embryos ineach ear. At 4 to 20 days, preferred at 6-15 days and more preferred at8-13 days and most preferred at 9-11 days after pollination, theimmature ears are harvested for transformation purpose. The haploidembryos are isolated based on the identification of the visible markergene in the inducer lines. For example, if the inducer contains a GFPgene or CRC gene driven by a promoter that allows the GFP or CRC geneexpression only in the embryos at the early developmental stage. Typicalpromoters that are useful include the maize oleosin promoter or maizeLec1 promoter etc. The haploid produced by this system is a maternalhaploid that has only one set of chromosomes from the female parent inthe embryo cells and has 3 sets of chromosomes in the endosperm cells,two of them from female parent and one of them from male parent. If theinducer line has a visible marker gene, such as GFP or CRC, this markergene will be included in the endosperm cells only, but not in the embryocells in the haploid kernels. By using this kind of visible marker,haploid embryos can be identified easily and can be isolated byrecognizing either the GFP negative or CRC negative embryos.

Haploid maize embryos from greenhouse or field grown plants arebombarded with a plasmid containing a gene of interest. The maizeembryos are isolated from ears 9-11 days after pollination using ascalpel. The ears are surface sterilized in 30% Clorox bleach plus 0.5%Micro detergent for 20 minutes, and rinsed two times with sterile water.The embryos are excised and placed embryo axis side down (scutellum sideup), 25 embryos per plate, on 560 Y medium for 4 hours and then alignedwithin the 2.5-cm target zone in preparation for bombardment.

A plasmid vector comprising a polynucleotide of interest operably linkedto a selected promoter is made. This plasmid DNA plus plasmid DNAcontaining a PAT selectable marker is precipitated onto 1.1 μm (averagediameter) tungsten pellets using a CaCl2 precipitation procedure asfollows: 100 μl prepared tungsten particles in water, 10 μl (1 μg) DNAin TrisEDTA buffer (1 μg total), 100 μl 2.5 M CaCl₂, 10 μl 0.1 Mspermidine.

Each reagent is added sequentially to the tungsten particle suspension,while maintained on the multitube vortexer. The final mixture issonicated briefly and allowed to incubate under constant vortexing for10 minutes. After the precipitation period, the tubes are centrifugedbriefly, liquid removed, washed with 500 μl 100% ethanol, andcentrifuged for 30 seconds. Again the liquid is removed, and 105 μl 100%ethanol is added to the final tungsten particle pellet. For particle gunbombardment, the tungsten/DNA particles are briefly sonicated and 10 μlspotted onto the center of each macrocarrier and allowed to dry about 2minutes before bombardment. The sample plates are bombarded at level #4in particle gun #HE34-1 or #HE34-2. All samples receive a single shot at650 PSI, with a total of ten aliquots taken from each tube of preparedparticles/DNA.

Following bombardment, the embryos are kept on 560 Y medium for 2 days,then transferred to 560 R selection medium containing 3 mg/literBialaphos, and subcultured every 2 weeks. After approximately 10 weeksof selection, selection-resistant callus clones are transferred to 288 Jmedium to initiate plant regeneration. Following somatic embryomaturation (2-4 weeks), well-developed somatic embryos are transferredto medium for germination and transferred to the lighted culture room.Approximately 7-10 days later, developing plantlets are transferred to272V hormone-free medium in tubes for 7-10 days until plantlets are wellestablished. Plants are then transferred to inserts in flats (equivalentto 2.5″ pot) containing potting soil and grown for 1 week in a growthchamber, subsequently grown an additional 1-2 weeks in the greenhouse,then transferred to classic 600 pots (1.6 gallon) and grown to maturity.Plants are monitored and scored for the genotype and/or phenotype ofinterest.

Bombardment medium (560 Y) comprises 4.0 g/l N6 basal salts (SIGMAC-1416), 1.0 ml/l Eriksson's Vitamin Mix (1000× SIGMA-1511), 0.5 mg/lthiamine HCl, 120.0 g/l sucrose, 1.0 mg/l 2,4-D, and 2.88 g/l L-proline(brought to volume with D-l H20 following adjustment to pH 5.8 withKOH); 2.0 g/l Gelrite (added after bringing to volume with D-l H20); and8.5 mg/l silver nitrate (added after sterilizing the medium and coolingto room temperature). Selection medium (560 R) comprises 4.0 g/l N6basal salts (SIGMA C-1416), 1.0 ml/l Eriksson's Vitamin Mix (1000×SIGMA-1511), 0.5 mg/l thiamine HCl, 30.0 g/l sucrose, and 2.0 mg/l 2,4-D(brought to volume with D-l H20 following adjustment to pH 5.8 withKOH); 3.0 g/l Gelrite (added after bringing to volume with D-l H20); and0.85 mg/l silver nitrate and 3.0 mg/l bialaphos(both added aftersterilizing the medium and cooling to room temperature).

Embryo-derived callus tissue is cultured on selection medium for about2-3 months. Putative stable transformed callus can be identified basedon the callus growth on herbicide-containing selection medium.Chromosome doubling can be performed at this stage or at the beginningof plant regeneration. Chromosome doubling agents, such as colchicine(0.01%-0.2%) or APM (5-225 μM) or Pronamide (0.5-20 μM) are added toeither callus selection medium (560 R) or plant regeneration medium (288J). Callus tissue is maintained on those media for 1 to a few days andthe samples of the treated calli are examined periodically to confirmchromosome doubling. In the alternative, the callus tissue can bemaintained in a chamber or a container in which N₂O (nitrous oxide) isprovided at 2-12 atmospheres for a few hours to a few days. Callussamples are examined to confirm chromosome doubling. Regular plantregeneration procedures are used following chromosome doubling.

Plant regeneration medium (288 J) comprises 4.3 g/l MS salts (GIBCO11117-074), 5.0 ml/l MS vitamins stock solution (0.100 g nicotinic acid,0.02 g/l thiamine HCL, 0.10 g/l pyridoxine HCL, and 0.40 g/l glycinebrought to volume with polished D-l H20) (Murashige and Skoog (1962)Physiol. Plant. 15:473), 100 mg/l myo-inositol, 0.5 mg/l zeatin, 60 g/lsucrose, and 1.0 ml/l of 0.1 mM abscisic acid (brought to volume withpolished D-l H20 after adjusting to pH 5.6); 3.0 g/l Gelrite (addedafter bringing to volume with D-l H20); and 1.0 mg/l indoleacetic acidand 3.0 mg/l bialaphos (added after sterilizing the medium and coolingto 60° C.). Hormone-free medium (272V) comprises 4.3 g/l MS salts (GIBCO11117-074), 5.0 ml/l MS vitamins stock solution (0.100 g/l nicotinicacid, 0.02 g/l thiamine HCL, 0.10 g/l pyridoxine HCL, and 0.40 g/lglycine brought to volume with polished D-l H20), 0.1 g/l myo-inositol,and 40.0 g/l sucrose (brought to volume with polished D-l H20 afteradjusting pH to 5.6); and 6 g/l bacto-agar (added after bringing tovolume with polished D-l H20), sterilized and cooled to 60° C.

Chromosomal doubling can also be performed in the regenerated plants,haploid seeds or the haploid seedlings germinated from haploid seeds.Similar doubling agents can be applied to these explants.

RepA can be used to increase chromosomal doubling efficiency. Haploidplant tissues (callus, seeds, seedlings etc.) containing the RepApolynucleotide are used in chromosomal doubling with the methodsdescribed above. The frequency of doubled haploids is increased severalfold.

RepA containing materials are compared with non-RepA materials forchromosomal doubling efficiency in corn haploid plants. Both types ofmaterials are grown in the same condition at the same time. However, thehaploid plants that contain RepA polynucleotide exhibit 60% chromosomaldoubling frequency versus non-RepA haploid plants which exhibit 26%chromosomal doubling frequency. The RepA containing doubled haploidplants produced 28 kernels per plants on the average and the non-RepAdoubled plants have produced 23 kernels per plants on the average.

Example 2 Agrobacterium-Mediated Transformation of Maize

For Agrobacterium-mediated transformation of maize the method of Zhao isemployed essentially as described in U.S. Pat. No. 5,981,840, thecontents of which are hereby incorporated by reference. Haploid embryos(preferably about 7-13 days after pollination) are isolated from maizeand the embryos are contacted with a suspension of Agrobacterium, wherethe bacteria are capable of transferring the nucleotide sequence(s) ofinterest to at least one cell of at least one of the embryos. In thisstep the embryos are typically immersed in an Agrobacterium suspensionfor the initiation of inoculation. Preferably, the Agrobacteriumsuspension contains 100 μM acetosyringone. The embryos are co-culturedfor a time with the Agrobacterium.

Generally the embryos are cultured on solid medium following theinfection step. Following this co-cultivation period an optional“resting” step lasting 6-7 days is contemplated. In this resting step,the embryos are incubated in the presence of at least one antibioticknown to inhibit the growth of Agrobacterium without the addition of aselective agent for plant transformants. Next, inoculated embryos arecultured on solid medium containing a selective agent and growingtransformed callus is recovered. The callus is then regenerated intoplants, and calli grown on selective medium are cultured on solid mediumto regenerate the plants.

Haploid immature embryos were isolated from maize Hi-II immature earsthat were pollinated with haploid inducer line RWS. These embryos wereas transformed by the method described in U.S. Pat. No. 5,981,840. Hi-IIimmature ears pollinated with haploid inducer line RWS contain bothdiploid embryos (averaged ˜87%) and haploid embryos (averaged ˜13%). Amarker gene was used that expressed at early stage of immature embryos(8-14 days after pollination) in the haploid inducer line RWS toidentify haploid immature embryos in Hi-II ears. Stably transformedhaploid embryos can be confirmed either by the transgene expression inthe transformed callus, regenerated plants and seeds etc. or bymolecular analysis, such as PCR, Southern blots and Northern blots etc.The data from 6 experiments are listed in Table 1 to demonstrate thecapability to transform haploid explants. The following constructs wereused for the transformation.

Ubi:GFP:pinII and 35 S:Bar:pinII were used for experiments 1, 2, and 5.

Nos:Lec1:pinII and Ubi:MO-PAT::GFP:pinII were used in experiments 3, 4,and 6. TABLE 1 Transformation of Maize Hi-II Haploid Embryos Total TotalTransformed Frequency Experiment embryo haploid haploid of haploid No.No. embryos embryos transformation 1 108  14 3 21% 2 84 11 2 18% 3 32  41 25% 4 93 12 6 50% 5 95 12 5 42% 6 120  16 7 44% Sum 532  69 24  35%

The method of chromosome doubling is essentially the same as forExample 1. The media used in plant regeneration is disclosed in U.S.Pat. No. 5,981,840.

Example 3 Transformation of Meristematic Tissue from Haploid Seeds

Transformation of meristematic tissue is disclosed in U.S. Pat. No.5,736,369. The method comprises particle bombardment of meristem tissueat a very early stage of development and the selective enhancement oftransgenic sectors toward genetic homogeneity in cell layers thatcontribute to germline transmission. Embryos are obtained as describedin Example 1 and incubated on maturation medium. The apical dome ofseveral embryos is disrupted prior to bombardment to force the meristemto reorganize and form new meristematic areas. Mechanical disruption isperformed by means of micromanipulation needles. Embryos are maintainedin the dark at 28° C. for seven days on maturation medium and thentransferred to 272K medium containing 150 mg/L Tobramycin sulfate. Theembryos will have multiple meristem formation with elongated cotyledons.The embryos are then incubated in light at 28° C. A chromosome doublingagent is used at the initiative stage of the novel meristems to doublechromosomes of the resulting transformed plants.

Example 4 Transformation of Somatic Tissue Derived from Haploid Seeds

Fertile plants have been developed from the leaf segments in a number ofmonocot species such as Orchard grass (Hanning and Conger 1982, Theor.Appl. Genet. 63:155-159; Conger et al. 1983, Science 221:850-851;Trigiano, et al. 1989, Bot. Gaz. 150:72-77), Maize (Chang, 1983, PlantCell Reports 2:183-185; Conger et al. 1987, Plant Cell Reports6:345-347; Wenzler and Meins 1986, Protoplasma 131:103-105; DebjaniSinha Ray and Ghosh 1990, Annals of Botany 66:497-500; Dolezelova et al.1992, Plant Cell Tissue and Organ Culture 31:215-221), Oat (Chen et al.1995, Plant Cell Reports 14:354-358; Chen et al. 1995, Plant CellReports 14:393-397), Sorghum (Wernocke and Brettel 1980, Nature287:138-139; Wernicke et al. 1982, Protolasma 111:53-62), and Wheat(Ahuja et al. 1982, Z. Pflanzenzuchtg 89:145-157).

Somatic tissues derived from seeds have been used for genetictransformation by both particle gun bombardment and Agrobacterium.Zarate et al. (1999, Biotechnology Lettes 21:997-1002) obtained stabletransformation via bombardment of Catharathus roseus plants throughadventitious organogenesis of buds isolated from germinated seeds. Zhonget al. (1996, Plant Physiol. 110:1097-1107) and U.S. Pat. No. 5,767,368(issued Jun. 6, 1998) disclosed the method of transformation of maize bybombarding meristem primordia which are derived from shoot apices.Reichert (U.S. Pat. No. 6,140,555, issued Oct. 31, 2000) transformednodal explants derived from germinated seeds by bombardment.Mahalakshmi-Akella and Khurana-Paramji disclosed Agrobacterium-mediatedtransformation of various tissues derived from mature seeds, such asleaf base, seedling and mature seeds in wheat (1996, Journal of PlantBiochemistry and Biotechnology 4:55-59). Methods described in Example 1and 3 are used for chromosome doubling.

Example 5 Identifying Haploids

Haploid cells, embryos, callus, plants etc. are identified with severalmethods, such as, by using Flow Cytometer to identify ploidy for alltissues, by chromosomal counting to identify ploidy for all tissues thatprovide dividing cells, and by measuring the length of guard cells orcounting the chloroplast numbers in guard cells of plant leaf tissues.Guard cells provide an easy and quick tool for ploidy assay at the plantstage.

Example 6 Screening of Transgenic Events

Transgenic plants are produced as described above. Because the resultingtransgenic events/plants are homozygous, all of the genes includingdominant and recessive genes are expressed in the plants. Carefullyevaluating the expression profile of the gene of interest and theinteraction of the gene with the whole genome at an early generation (T0and/or T1) will allow researchers to discard unnecessary events andfocus on the candidate events at an early stage. If these doubledhaploid lines are suitable for hybrid formation they can be useddirectly in hybrid yield testing as top crosses or as putative finalproducts.

Example 7 Transforming an Inducer Line with a Marker Gene to IdentifyHaploid Immature Embryos at Early Stage

Haploid inducer lines, such as RWS, KEMS, ZMS or KMS, do not have amarker gene that can be expressed in the early embryo development stage,such as 8-12 days after pollination. However, transforming embryos inmost plants, such as corn, sorghum, wheat, barley and rice etc., usuallyrequires embryos at an early development stage. The marker gene(s) usedin the current inducer lines does not express well at early embryodevelopment stage. To identify haploid immature embryos at the beginningof transformation process is useful to the efficient development of newtechnology for haploid transformation or to modify the existingtechnology to favor haploid targets.

The Lec1 promoter can provide expression during early embryo developmentand during the callus stage of plant regeneration. The lec1 promoter isdescribed in US PCT Application No. U.S. Ser. No. 01/44732 filed Nov.20, 2001, the disclosure of which is incorporated herein by reference.

An inducer line, such as RWS in corn, is transformed with the Lec1promoter driving a marker gene, such as GFP, YFP, CFP or CRC etc. Thisinducer line is used as the male parent to pollinate any other genotypesof corn plants. The resulting ears contain both haploid and diploidkernels. The Lec1 promoter is an early active promoter in immatureembryos. The gene driven by the Lec1 promoter can be expressed as earlyas 7 days after pollination and can last until 15 days afterpollination. In addition, the gene driven by the Lec1 promoter isexpressed only in the embryos, and not in the endosperm. The haploidkernels derived from a genotype pollinated by the inducer line containthe inducer genomic set in their endosperm only, but not in theirembryos. On the other hand, the diploid kernels from this cross containthe inducer genomic set in both the embryo and the endosperm. Theresulting haploid embryos do not contain the marker gene and the diploidembryos do contain the marker gene. In this way the haploid embryos canbe easily identified at the beginning of transformation. Also, anymarker gene used in the inducer line transformation will not be presentin the haploid embryos and will not affect the resulting doubled haploidtransformants.

Example 8 Transforming an Inducer Line with an Inducible Seed LethalGene

In the present invention haploid inducer lines, such as RWS, KEMS, ZMSor KMS, can be transformed with a lethal gene that is expressedspecifically in embryos of the mature seeds. The expression of thelethal gene is controlled with an inducible system. Because theexpression of the lethal gene is controlled by an inducible promoter, itcannot express in the inducer lines when the inducing agent is notpresent. The seeds of the inducer lines can be germinated as normalseeds. After crossing the inducer line and the female parent the embryosof all the diploid seeds contain the inducible lethal gene, but theembryos of the haploid seeds do not contain the lethal gene. By inducingthe expression of the lethal gene when the F1 seeds germinate, thediploid F1 seeds cannot germinate due to expression of the lethal genein their embryos. However, all of the haploid seeds can germinatenormally because they do not contain the lethal gene in their embryos.All of the germinating seedlings are haploid.

The plant cell is transformed with a DNA sequence whose expression islethal to the plant cell and an inducible promoter which contains areceptor binding site. When the receptor binds to the binding site, thepromoter is functional and results in the expression of the lethal gene.The plant cell is also transformed with a second DNA sequence whoseexpression produces a precursor of the receptor which can interact withan external agent to form a functional receptor. The functional receptorthen binds to the receptor binding site to induce the expression of thelethal gene.

An alternative way to induce the expression of a lethal gene is bytransforming a lethal gene into a plant cell, wherein the expression ofthe lethal gene is controlled by a plant-active promoter. The lethalgene and the promoter are linked to each other, but separated by ablocking sequence that is flanked by specific excision sequences. Thepresence of the blocking sequence prevents the expression of the lethalgene in the plant cell. The plant cell is also transformed with a secondgene that encodes a recombinase specific for the excision sequencesflanking the blocking sequence of the lethal gene. The second gene isoperably linked to an inducible promoter that contains a receptorbinding site. When the receptor binds to the binding site, the promoteris functional and results in the expression of the recombinase. Theplant cell is transformed with a third gene whose expression producesthe precursor of the receptor that can interact with an external agentto form a functional receptor. The functional receptor binds to thebinding site to produce expression of the recombinase. Expression of therecombinase results in excision of the blocking sequence between theplant-active promoter and the lethal gene and results in expression ofthe lethal gene in the plant cell. In this case, if the induciblepromoter is not 100% tightly-induced by adding the external agent, thepromoter leaks at a low level which is not sufficient to inducerecombinase activity, it should not be lethal to the plant cell.

A third method to produce an inducible system comprises transforming alethal gene into a plant cell. The expression of the lethal gene iscontrolled by an inducible promoter. The lethal gene and the induciblepromoter are linked to each other, but separated by a blocking sequencethat is flanked by specific excision sequences. The presence of theblocking sequence prevents the expression of the lethal gene in theplant cell. The plant cell is also transformed with a second gene thatencodes a recombinase specific for the specific excision sequencesflanking the blocking sequences of the lethal gene and a secondinducible promoter that contains a receptor binding site. Induction ofthe second inducible promoter is different from the first induciblepromoter as different inducing agents are used. When the receptor bindsto the binding site, the promoter is functional and results in theexpression of the recombinase. The plant cell is transformed with athird gene whose expression produces a precursor of the receptor thatcan interact with an external agent to form a functional receptor andthe functional receptor binds to the binding site to cause expression ofthe recombinase. The expression of the recombinase results in theexcision of the blocking sequence between the first inducible promoterand the lethal gene and by adding the appropriate inducing agent toinduce this promoter function, the lethal gene can be expressed in theplant cell. In this case, if both inducible promoters are not 100%tightly-induced by adding the external agent, the effect of the promoterleaking will be significantly diluted.

In the above methods, the inducing agent can be mixed with a seedcoating mix for F1 seeds. After planting the seeds in the field, thediploid seeds cannot germinate due to the induction of the expression ofthe lethal gene.

A typical inducible receptor binding sequence is ERE (EstrogenResponsive Element) (Klein-Hitpass et al. 1988, Nucleic Acids Res.16:647-663; Bruce et al. 2000, The Plant Cell, 12:65-79). Typicalinducing agents are estradiols (β-estradiol-17-[β-D-glucuronide] orβ-estradiol-3-[β-D-glucuronide or 17α-ethylnylestradiol for ERE) orsafener for CAS.

Typical inducible promoters are In-2 promoter (Hershey et al. WO9011361) and tetracycline-inducible promoter (Bellingcampi et al.,1996,Plant Cell 8:477-487; De-Veylder-Lieven et al., 2000, Journal ofExperimental Botany 51:1647-1653), and typical inducing agents aresafener (for In-2 promoter) and tetracycline.

Typical lethal genes are DAM (DAM Methylase gene) or RIP (ribosomalinhibitor protein gene). Typical embryo specific promoters are gib-1 orOleosin. Typical recombinase genes are FLP and CRE and typical flankingsequences are to FRT and LOX.

Example 9 Providing a Growth-Stimulating Gene Increases the Frequency ofHaploid Embryos after Crossing the Inducer-Line to any Maize Genotype

There are two variations on this method.

A. In the first, the genotype of interest (i.e. PHN46) is transformedwith a growth-stimulating polynucleotide (GS) operably linked to apromoter that drives expression in the embryo. For example, a cassettecontaining LTP2::LEC1::pinII is introduced into PHN46. T0 plants areregenerated and are selfed to produce seed that are LEC1/LEC1homozygous. These seed are germinated, the plants grown to maturity, andpollinated with the haploid-inducer line. For comparison, wild-type(non-transformed) PHN46 seed is also planted and pollinated with thehaploid inducer. For ears on wild type PHN46 plants, the frequency ofkernels containing haploid embryos ranges between 5-10%. For earsdeveloping on LEC1/LEC1 PHN46 plants, the frequency of kernels thatcontain haploid embryos is expected to be higher, for example rangingbetween 10-20%.

B. In the second variation on this method, the inducer line istransformed with an expression cassette containing an endosperm-specificpromoter, a GS gene, and an endosperm-specific 3′ sequence. For example,GZ::PSK::GZ is transformed into the haploid-inducer line. Plants areregenerated and selfed to produce PSK/PSK homozygous seed. The PSK/PSKhaploid-inducer seed is planted, grown to maturity and used to pollinateears from PHN46. For comparison, non-transgenic haploid-inducer seedsare also planted, and the resultant plants used to pollinate ears fromPHN46. For PHN46 ears pollinated with non-transgenic haploid-inducerpollen, the frequency of kernels containing haploid embryos rangesbetween 5-10%. For PHN46 ears pollinated using pollen from transgenicPSK/PSK haploid-inducer plants, the frequency of kernels that containhaploid embryos is expected to be higher, for example ranging between10-20%.

Example 10 Providing a Growth-Stimulating Gene Increases the Frequencyof Haploid Embryo Transformation

Again, there are two variations on this method.

A. In the first, any given genotype (i.e. PHN46) is transformed with agrowth-stimulating (GS) gene operably linked a promoter that drivesexpression in the embryo. For example, a cassette containingLec1::RepA::Lec1 is introduced into PHN46. T0 plants are regenerated andselfed to produce seed that are RepA/RepA homozygous. These seed aregerminated, the plants grown to maturity, and pollinated with thehaploid-inducer line. For comparison, wild-type (non-transformed) PHN46seed is also planted and pollinated with the haploid inducer. For PHN46RepA/RepA transgenic ears pollinated with haploid-inducer pollen, theAgrobacterium-mediated transformation frequency is expected to fall inthe range between 8-20%. For wild type non-transformed PHN46 earspollinated using the haploid-inducer line, the Agrobacterium-mediatedtransformation frequency ranges between 2-4%.

B. In the second variation on this method, the inducer line istransformed with an expression cassette containing an endosperm-specificpromoter, a GS gene, and an endosperm-specific 3′ sequence. For example,GZ::PSK::GZ is transformed into the haploid-inducer line (the GZpromoter is disclosed in Ueda et al. (1994) Mol Cell Biol14(7):4350-4359). Plants are regenerated and selfed to produce PSK/PSKhomozygous seed. The PSK/PSK haploid-inducer seed is planted, grown tomaturity and used to pollinate ears from PHN46. For comparison,non-transgenic haploid-inducer seeds are also planted, and the resultantplants used to pollinate ears from PHN46. For PHN46 ears pollinated withnon-transgenic haploid-inducer pollen, the Agrobacterium-mediatedtransformation frequency ranges between 2-4%. For PHN46 ears pollinatedusing pollen from a homozygous transgenic (PSK/PSK) haploid-inducerplant, the Agrobacterium-mediated transformation frequency is expectedto range between 8-20%.

Example 11 Providing a Growth-Stimulation Gene Increases the Frequencyof Chromosome Doubling in Haploid Cells

The genotype of interest is transformed with a GS gene, pollinated withthe haploid inducer line, and the resultant haploid progeny will havehigher chromosome doubling rates (induced either in the callus stage orin germinating seedlings) relative to non-transformed material from thesame genotype.

General note: Examples 9 and 10 could theoretically utilize any of thepotential GS genes listed. Example 11 covers either over-expression ofGS genes that stimulate the cell cycle such as Cyclin A, Cyclin B,Cyclin C, Cyclin D, Cyclin E, Cyclin F, Cyclin G, and Cyclin H; Pin1;E2F; Cdc25; RepA, or to suppressing activity of cell cycle repressorssuch as Rb, CKI, prohibitin, or wee1. Suppressing activity could involveantisense, hairpins, sense co-suppression, over-expressing adominant-negative mutant, expressing antibodies raised against theprotein, etc.

1. A method of making a doubled haploid maize plant comprising: a)contacting a maize haploid embryo with a chromosome doubling agent toproduce a doubled haploid embryo; b) generating a doubled haploid maizeplant from said doubled haploid embryo.
 2. The method of claim 1 whereinsaid embryo is produced by crossing a female plant with a male inducerline.
 3. The method of claim 2 wherein the inducer line contains ascorable marker gene.
 4. The method of claim 3 wherein said marker geneis selected from the group consisting of an anthocyanin gene, R-nj, GFP,and lec1 promoter driving CRC.
 5. The method of claim 1 wherein saiddoubling agent is selected from the group consisting of colchicines,nitrous oxide, and pronamide.
 6. The method of claim 1 furthercomprising transforming said haploid embryo before contacting saidhaploid embryo with said doubling agent.
 7. A method of making a doubledhaploid maize plant comprising: a) generating callus from a maizehaploid embryo b) contacting said callus with a chromosome doublingagent; c) generating a doubled haploid somatic embryo from said callus;d) generating a doubled haploid maize plant from said doubled haploidsomatic embryo.
 8. The method of claim 7 wherein said immature embryo isproduced by crossing a female plant with a male inducer line.
 9. Themethod of claim 8 wherein the inducer line contains a scorable markergene.
 10. The method of claim 9 wherein said marker gene is selectedfrom the group consisting of an anthocyanin gene, R-nj, GFP, and lec1promoter driving CRC.
 11. The method of claim 7 wherein said doublingagent is selected from the group consisting of colchicine, nitrousoxide, and pronamide.
 12. The method of claim 7 further comprisingtransforming said callus before contacting said callus with saiddoubling agent.
 13. A method of making a doubled haploid maize cellcomprising: contacting a maize haploid embryo with a chromosome doublingagent to produce a doubled haploid cell.
 14. The method of claim 13wherein said embryo is produced by crossing a female plant with a maleinducer line.
 15. The method of claim 14 wherein the inducer linecontains a scorable marker gene.
 16. The method of claim 15 wherein saidmarker gene is selected from the group consisting of an anthocyaningene, R-nj, GFP, and lec1 promoter driving CRC.
 17. The method of claim13 wherein said doubling agent is selected from the group consisting ofcolchicine, nitrous oxide, and pronamide.
 18. A method of making adoubled haploid maize embryo comprising: contacting a maize haploidembryo with a chromosome doubling agent to produce a doubled haploidembryo.
 19. The method of claim 18 wherein said embryo is produced bycrossing a female plant with a male inducer line.
 20. The method ofclaim 19 wherein the inducer line contains a scorable marker gene. 21.The method of claim 20 wherein said marker gene is selected from thegroup consisting of an anthocyanin gene, R-nj, GFP, and lec1 promoterdriving CRC.
 22. The method of claim 18 wherein said doubling agent isselected from the group consisting of colchicine, nitrous oxide, andpronamide.